Mitochondrial bioenergetics is crucial for cell survival and death. The bioenergetic maintenance primarily depends on the integrity of mitochondrial membranes. The impermeable nature of the mitochondrial inner membrane sets the stage for redox reactions to generate ATP. Mitochondria also participate in cytosolic Ca2+ phenotype via rapid Ca2+ buffering. There are two sides to the effects of Ca2+ on mitochondrial function. Under physiological conditions, Ca2+ is beneficial for mitochondrial function to stimulate oxidation-phosphorylation and ATP synthesis. It is questionable whether these effects remain the same under pathological conditions when mitochondrial Ca2+ ([Ca2+]m) overload occurs. While [Ca2+]m signaling is crucial for both physiological and pathological processes, molecules that facilitate [Ca2+]m uptake remain unclear. [Ca2+]m buffering is exquisitely controlled by inner mitochondrial membrane transporters, exchangers and uniporter. Several proteins have been implicated to participate in [Ca2+]m uptake, including LETM1, MICU1 and MCU. Our targeted RNAi screen identified a mitochondrial inner membrane protein, Mitochondrial Ca2+ Uniporter Regulator 1 (MCUR1) that augments [Ca2+]m uptake. MCUR1 silencing abrogates [Ca2+]m uptake under normal mitochondrial membrane potential. Our results demonstrate that MCUR1 interacts with the Ru360 sensitive core component of the mitochondrial uniporter complex, Mitochondrial Ca2+ Uniporter (MCU). Based on our recent discovery, we hypothesize that MCUR1 promotes MCU-dependent [Ca2+]m overload during I/R injury, triggering mitochondrial membrane depolarization, that results in bioenergetic collapse and mitochondrial dysfunction. This proposal applies RNAi technology, mutagenesis of MCUR1 and MCU channel, biochemical, state-of-the-art imaging and an animal model system to understand how MCUR1 elicits cardiomyocyte [Ca2+]m uptake. Based on our recent identification of MCUR1 as a regulator of the uniporter complex, here in Aim 1, we will characterize the MCUR1 role in cardiomyocyte [Ca2+]m uptake, critical regions of MCUR1-MCU interaction and transcriptional regulation of MCUR1. In Aim 2 we will investigate how MCUR1 controls mitochondrial bioenergetics, ROS production and autophagy. Finally, in Aim 3 we will apply cardiac ischemia/reperfusion in vivo murine model studies to show that knockdown of MCUR1 ameliorates I/R-induced mitochondrial dysfunction and cardiomyocyte damage. Overall, the results of these studies will advance our understanding of how MCU activity is augmented under pathophysiological conditions, and suggest new strategies for controlling [Ca2+]m influx as a new treatment for cardiovascular diseases.